Vitamin b2 detection by mass spectrometry

ABSTRACT

Methods are described for measuring the amount of a vitamin B2 in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying vitamin B2 in a sample utilizing on-line extraction methods coupled with tandem mass spectrometric techniques.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.12/365,080, filed Feb. 3, 2009, which claims priority from ProvisionalApplication U.S. Application 61/138,909, filed Dec. 18, 2008, the entirecontents of each are incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of vitamin B2. Ina particular aspect, the invention relates to methods for quantitativemeasurement of vitamin B2 by HPLC-tandem mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Vitamin B2 (also known as riboflavin) is one of eight water-soluble Bvitamins. It is the central component of the cofactors flavin adeninedinucleotide (FAD) and flavin mononucleotide (FMN), and is thereforerequired by all flavoproteins. As such, vitamin B2 is required for awide variety of cellular processes. Like the other B vitamins, it playsa key role in energy metabolism, and is required for the metabolism offats, ketone bodies, carbohydrates, and proteins. Vitamin B2 is alsorequired for the health of the mucus membranes in the digestive tractand helps with the absorption of iron and Vitamin B6.

Riboflavin is continuously excreted in the urine of healthy individuals,making deficiency relatively common when dietary intake is insufficient.However, a deficiency of riboflavin may also be due to secondary causes,i.e., the result of conditions that affect absorption in the intestine,the body's inability to use vitamin B2, or an increase in the excretionof vitamin B2 from the body.

In humans, signs and symptoms of riboflavin deficiency (ariboflavinosis)include cracked and red lips, inflammation of the lining of mouth andtongue, mouth ulcers, cracks at the corners of the mouth (angularcheilitis), and sore throat. A deficiency may also cause dry and scalingskin, fluid in the mucous membranes, and iron-deficiency anemia. Theeyes may also become bloodshot, itchy, watery and sensitive to brightlight.

Riboflavin deficiency is classically associated with theoral-ocular-genital syndrome. Angular cheilitis, photophobia, andscrotal dermatitis are the classic remembered signs.

Methods have been reported for extracting vitamin B from food/drinksamples or dietary supplements. See, e.g., Leporati, A., et al.,Analytica Chimica Acta 2004, 531:87-95; Zougagh M, et al.,Electrophoresis 2008, 29:3213-9; Chen, P., et al., Anal Bioanal Chem2007, 387:2441-8; Aranda, et al., J. Chromatrogr. A 2006, 1131:253-60;Gentili, et al., Rapid Commun. Mass Spectrom. 2008, 22:2029-43; andGrant, D., et al., Anal Bioanal Chem 2008, 391:2811-2818. Additionally,various mass spectrometric techniques for measuring vitamin B2 in asample have been reported. See, e.g., Leporati, A., et al., AnalyticaChimica Acta 2004, 531:87-95; Guo, et al., J. Chromatographic Science2006, 44:552-6; Midttun, Ø., et al., Clin. Chem. 2005, 51:1206-16; Chen,P., et al., Anal Bioanal Chem 2007, 387:2441-8; and Grant, D., et al.,Anal Bioanal Chem 2008, 391:2811-2818.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence oramount of vitamin B2 in a sample by tandem mass spectrometry.Preferably, the methods of the invention include a solid phaseextraction method coupled with high performance liquid chromatography.

In one aspect, methods are provided for determining the amount ofvitamin B2 in a biological sample. In some embodiments, the methodsinclude: (a) subjecting the sample, purified by solid phase extraction(SPE), to ionization under conditions suitable to produce one or moreions detectable by mass spectrometry; (b) determining the amount of oneor more ions by tandem mass spectrometry; and (c) using the amount ofthe one or more ions to determine the amount of vitamin B2 in thesample. In some embodiments, SPE comprises turbulent flow liquidchromatography (TFLC). In some embodiments, SPE and tandem massspectrometry are conducted with on-line processing. In some embodiments,the sample purified by SPE has been further purified by high performanceliquid chromatography (HPLC) prior to ionization; preferably withon-line processing. In some embodiments, the one or more ions detectedby tandem mass spectrometry are selected from the group consisting ofions with mass to charge ratio of 377.2±0.5 and 243.2±0.5. The featuresof the embodiments listed above may be combined without limitation foruse in methods of the present invention.

In other embodiments, methods for determining the amount of vitamin B2in a sample include: (a) subjecting the sample, purified by turbulentflow chromatography (TFLC) and high performance liquid chromatography(HPLC), to ionization under conditions suitable to produce one or moreions detectable by mass spectrometry; wherein one or more ionsdetectable by mass spectrometry comprise one or more ions from the groupconsisting of ions with a mass to charge ratio of 377.2±0.5 and243.2±0.5; (b) determining the amount of one or more ions by tandem massspectrometry; and (c) using the amount of the one or more ions todetermine the amount of vitamin B2 in the sample. In some embodiments,the TFLC and HPLC are conducted as on-line processing of the sampleprior to mass spectrometry. In some embodiments, the one or more ionsdetectable by mass spectrometry comprise a parent ion with a mass tocharge ratio of 377.2±0.5 and a fragment ion with a mass to charge ratioof 243.2±0.5. The features of the embodiments listed above may becombined without limitation for use in methods of the present invention.

Methods of the present invention may involve the combination of liquidchromatography with mass spectrometry. In preferred embodiments, theliquid chromatography is TFLC. One preferred embodiment utilizes TFLC incombination with one or more purification methods such as for exampleSPE (e.g., TFLC) and/or protein precipitation and filtration, to purifyan analyte in a sample. In some embodiments, at least one purificationstep and mass spectrometric analysis is conducted in an on-line fashion.In another preferred embodiment, the mass spectrometry is tandem massspectrometry (MS/MS).

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain preferred embodiments, vitamin B2 ismeasured using ESI in positive ion mode.

In some preferred embodiments, vitamin B2 ions detectable in a massspectrometer are selected from the group consisting of positive ionswith a mass/charge ratio (m/z) of 377.20±0.50 and 243.20±0.50. Inparticularly preferred embodiments, a vitamin B2 precursor ion has m/zof 377.20±0.50, and a fragment ion has m/z of 243.20±0.50.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the analyte ofinterest and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry.Preferably, the internal standard is ¹³C, ¹⁵N₂-vitamin B2. In theseembodiments, ¹³C, ¹⁵N₂-vitamin B2 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with amass/charge ratio (m/z) of 380.20±0.50 and 243.20±0.50. In particularlypreferred embodiments, a ¹³C, ¹⁵N₂-vitamin B2 precursor ion has m/z of380.20±0.50, and a fragment ion has m/z of 243.20±0.50. In theseembodiments, the presence or amount of ions generated from the analyteof interest may be related to the presence of amount of analyte ofinterest in the sample.

In other embodiments, the amount of the vitamin B2 ion or ions may bedetermined by comparison to one or more external reference standards.Exemplary external reference standards include blank plasma or serumspiked with vitamin B2 or ¹³C, ¹⁵N₂-vitamin B2.

In certain preferred embodiments, the limit of quantitation (LOQ) ofvitamin B2 is within the range of 5 nmol/L to 25 nmol/L, inclusive;preferably within the range of 5 nmol/L to 20 nmol/L, inclusive;preferably within the range of 5 nmol/L to 15 nmol/L, inclusive;preferably within the range of 5 nmol/L to 10 nmol/L, inclusive;preferably about 5 nmol/L.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. In preferred embodiments, the samplecomprises a body fluid sample; preferably plasma or serum.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit column exhibit strong cation exchange andhydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about 5μm in diameter.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show exemplary chromatograms for vitamin B2 and ¹³C,¹⁵N₂-vitamin B2 (internal standard), respectively. Details are discussedin Example 3.

FIG. 2 shows a plot of the coefficient of variation and accuracy ofassays of four standards used to determine the lower limit ofquantitation of the vitamin B2 assay. Details are discussed in Example5.

FIG. 3 shows plots of the linearity of quantitation of vitamin B2 inserially diluted stock samples for three channels of a four channelTFLC-MS/MS system conducted over two days. Details are described inExample 6.

FIGS. 4A-C show the correlation of determination of vitamin B2 in serumand vitamin B2 in EDTA plasma by TFLC-MS/MS determination. Details aredescribed in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of vitamin B2 in asample. More specifically, mass spectrometric methods are described fordetecting and quantifying vitamin B2 in a sample. The methods mayutilize turbulent flow liquid chromatography (TFLC), to perform apurification of selected analytes, combined with methods of massspectrometry (MS), thereby providing a high-throughput assay system fordetecting and quantifying vitamin B2 in a sample. The preferredembodiments are particularly well suited for application in largeclinical laboratories for automated vitamin B2 quantification assay.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma and serum; most preferably EDTAplasma. Such samples may be obtained, for example, from a patient; thatis, a living person, male or female, presenting oneself in a clinicalsetting for diagnosis, prognosis, or treatment of a disease orcondition.

The present invention also contemplates kits for a vitamin B2quantitation assay. A kit for a vitamin B2 quantitation assay mayinclude a kit comprising the compositions provided herein. For example,a kit may include packaging material and measured amounts of anisotopically labeled internal standard, in amounts sufficient for atleast one assay. Typically, the kits will also include instructionsrecorded in a tangible form (e.g., contained on paper or an electronicmedium) for using the packaged reagents for use in a vitamin B2quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix, provided that vitamin B2 is essentially absent. Commerciallyavailable EDTA plasma often contains a significant amount of Vitamin B2;thus in preferred embodiments, blank human serum (without vitamin B2,such as Biocell Laboratories, Inc., Cat. No. 1131-00) is used forcalibration and QC pools.

Sample Preparation for Mass Spectrometric Analysis

Typically, frozen test samples (including controls) are thawed rapidlyand kept protected from light exposure to minimize vitamin B2degradation. Internal standard may be added to the test samples oncethey are thawed.

In preparation for mass spectrometric analysis, vitamin B2 may beenriched relative to one or more other components in the sample (e.g.protein) by various methods known in the art, including for example,liquid chromatography, filtration, centrifugation, thin layerchromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate or methanolextraction, and the use of chaotropic agents or any combination of theabove or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving vitamin B2 in the supernatant. Thesamples may be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, the use of protein precipitation such as forexample, formic acid protein precipitation, may obviate the need forTFLC or other on-line extraction prior to mass spectrometry or HPLC andmass spectrometry.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a partitionprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with vitamin B2. The chromatographic columntypically includes a medium (i.e., a packing material) to facilitateseparation of chemical moieties (i.e., fractionation). The medium mayinclude minute particles. The particles typically include a bondedsurface that interacts with the various chemical moieties to facilitateseparation of the chemical moieties. One suitable bonded surface is ahydrophobic bonded surface such as an alkyl bonded or a cyano bondedsurface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18bonded alkyl groups. In preferred embodiments, the column is a C-18column. The chromatographic column includes an inlet port for receivinga sample and an outlet port for discharging an effluent that includesthe fractionated sample. The sample may be supplied to the inlet portdirectly, or from a SPE column, such as an on-line TFLC column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a polar embeddedanalytical column chromatographic system. In certain preferredembodiments, a polar embedded C-18 analytical column (e.g., an AtlantisT3 analytical column from Waters Corp. (5 μm particle size, 50×4.6 mm),or equivalent) is used. In certain preferred embodiments, HPLC and/orTFLC are performed using HPLC Grade acetonitrile, water, 0.1% aqueousformic acid, 0.1% formic acid in acetonitrile, and a solution ofacetonitrile, water, 0.5% ammonium formate, and 0.1% formic acid asmobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of vitamin B2prior to mass spectrometry. In such embodiments, samples may beextracted using a TFLC column which captures the analyte, then elutedand chromatographed on a second TFLC column or on an analytical HPLCcolumn. For example, sample extraction with a TFLC extraction cartridgemay be accomplished with a large particle size (50 μm) packed column.Sample eluted off of this column may then be transferred to an HPLCanalytical column for further purification prior to mass spectrometry.In preferred embodiments, a mixed mode material with both strong cationexchange and reversed phase binding capacity (e.g., a Cyclone MCX columnfrom Cohesive Technologies, Inc. (60 μm particle size, 1.0×50 mm), orequivalent) is used. Because the steps involved in these chromatographyprocedures may be linked in an automated fashion, the requirement foroperator involvement during the purification of the analyte can beminimized. This feature may result in savings of time and costs, andeliminate the opportunity for operator error.

Detection and Quantitation by Mass Spectrometry

In various embodiments, vitamin B2 present in a test sample may beionized by any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),fast atom bombardment (FAB), liquid secondary ionization (LSI), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP) andparticle beam ionization. The skilled artisan will understand that thechoice of ionization method may be determined based on the analyte to bemeasured, type of sample, the type of detector, the choice of positiveversus negative mode, etc.

Vitamin B2 may be ionized in positive or negative mode. In preferredembodiments, vitamin B2 is ionized by ESI in positive mode. In relatedpreferred embodiments, vitamin B2 ions are in a gaseous state and theinert collision gas is argon or nitrogen; preferably argon.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, ions may be detected using a scanningmode, e.g., multiple reaction monitoring (MRM) or selected reactionmonitoring (SRM). Preferably, the mass-to-charge ratio is determinedusing a quadrupole analyzer. For example, in a “quadrupole” or“quadrupole ion trap” instrument, ions in an oscillating radio frequencyfield experience a force proportional to the DC potential appliedbetween electrodes, the amplitude of the RF signal, and the mass/chargeratio. The voltage and amplitude may be selected so that only ionshaving a particular mass/charge ratio travel the length of thequadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 2000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of vitamin B2.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, in preferred embodiments isotopicallylabeled vitamin B2 (e.g., ¹³C, ¹⁵N₂-vitamin B2) may be used as aninternal standard. Numerous other methods for relating the amount of anion to the amount of the original molecule will be well known to thoseof ordinary skill in the art.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium,¹³C, and ¹⁵N. For example, ¹³C, ¹⁵N₂-vitamin B2 has a mass about threeunits higher than vitamin B2. The isotopic label can be incorporated atone or more positions in the molecule and one or more kinds of isotopiclabels can be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, vitamin B2 in a sample isdetected and/or quantified using MS/MS as follows. The samples aresubjected to liquid chromatography, preferably TFLC followed by HPLC;the flow of liquid solvent from a chromatographic column enters theheated nebulizer interface of an MS/MS analyzer; and the solvent/analytemixture is converted to vapor in the heated charged tubing of theinterface. During these processes, the analyte (i.e., vitamin B2) isanalyzed. The ions, e.g. precursor ions, pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions (i.e., selection of“precursor” and “fragment” ions in Q1 and Q3, respectively) based ontheir mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collisioncell, where ions are fragmented. The first quadrupole of the massspectrometer (Q1) selects for molecules with the mass to charge ratiosof vitamin B2. Precursor ions with the correct mass/charge ratios areallowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. The fragment ions generatedare passed into quadrupole 3 (Q3), where the fragment ions of vitamin B2are selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of vitamin B2 that maybe used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of vitamin B2. As described above, the relative abundance of agiven ion may be converted into an absolute amount of the originalanalyte using calibration standard curves based on peaks of one or moreions of an internal molecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods. In particular,the following Examples demonstrate quantitation of vitamin B2 by massspectrometry with the use of ¹³C, ¹⁵N₂-vitamin B2 as an internalstandard. The use of ¹³C, ¹⁵N₂-vitamin B2 as an internal standard is notmeant to be limiting in any way. Any appropriate chemical species,easily determined by one in the art, may be used as an internal standardfor vitamin B2 quantitation.

EXAMPLES Example 1 Reagent and Sample Preparation

Two internal standard solutions were prepared with ¹³C, ¹⁵N₂-vitamin B2(Moravek Biomedicals, Inc., custom made, or equivalent). A ¹³C,¹⁵N₂-vitamin B2 internal standard stock solution of 300 μmol/L ¹³C,¹⁵N₂-vitamin B2 in water was prepared. A 500 μL portion of this solutionwas then diluted to 1 L with water to prepare a ¹³C, ¹⁵N₂-vitamin B2internal standard working solution of 150 nmol/L.

Three calibrant solutions were prepared with vitamin B2 (U.S.Pharmacopia, Cat. No. 1603006, or equivalent). First, a vitamin B2calibrant stock solution of about 300 μmol/L vitamin B2 in water wasprepared. 10 mL of this solution was then diluted with about 90 mL waterto prepare a vitamin B2 intermediate calibrant solution of about 30μmol/L. 2.5 mL of the intermediate calibrant solution was then added to497.5 mL of blank human serum (Biocell Laboratories, Inc., Cat. No.1131-00, or equivalent) to prepare a vitamin B2 calibrant workingsolution of 150 nmol/L.

Portions of the 150 nmol/L vitamin B2 calibrant working solution werethen diluted with blank serum to prepare aliquots at 120 nmol/L, 75nmol/L, 60 nmol/L, 30 nmol/L, 15 nmol/L, and 7.5 nmol/L vitamin B2.

Plasma samples were prepared by collecting blood specimens in EDTAVacutainer tubes and protected from light. The collected specimens werethen frozen for storage and/or transport.

In preparation for analysis, frozen specimens were allowed to thaw andcentrifuged at 4000 rpm and 5° C. for 10 minutes. A 200 μL aliquot ofthe top layer of each centrifuged specimen was then transferred to awell in a 96 well sample tray. 200 μL aliquots of a blank and calibrantsat 150 nmol/L, 120 nmol/L, 75 nmol/L, 60 nmol/L, 30 nmol/L, 15 nmol/L,and 7.5 nmol/L were also transferred to wells in the sample tray.

800 μL of the 150 nmol/L internal standard working solution was thenadded to each sample, blank, and calibrant in the tray.

Example 2 Extraction of Vitamin B2 from Samples Using LiquidChromatography

Sample injection was performed with a Cohesive Technologies Aria TLX-4TFLC system using Aria OS V 1.5.1 or newer software. This system allowsfor simultaneous chromatography of up to four samples. In the Examplesdescribed below, up to three channels were used for simultaneouschromatography of three samples.

For each channel, the TFLC system automatically injected 60 μL of theabove prepared samples into a Cyclone MCX column (60 μm particle size,1.0×50 mm, from Cohesive Technologies, Inc.) packed with largeparticles. The samples were loaded at a high flow rate (2.0 mL/min,loading reagent 100% water) to create turbulence inside the extractioncolumn. This turbulence ensured optimized binding of vitamin B2 to thelarge particles in the column and the passage of residual protein anddebris to waste.

Following loading, the sample was eluted off to the analytical column(polar embedded C-18 Atlantis T3 analytical column from Waters Corp. (5μm particle size, 50×4.6 mm), or equivalent) with an eluting solvent of20% acetonitrile, 79.4% water, 0.5% ammonium formate, and 0.1% formicacid. The HPLC gradient was applied to the analytical column, toseparate vitamin B2 from other analytes contained in the sample. Mobilephase A was 0.1% formic acid in water and mobile phase B was 0.1% formicacid in acetonitrile. The HPLC gradient started with a 30% organicgradient which was ramped to 50% in approximately 30 seconds, thenramped to 100% in approximately 50 seconds.

The separated samples were then subjected to MS/MS for quantitation ofvitamin B2.

Example 3 Detection and Quantitation of vitamin B2 by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs, all fromThermo Electron, were used in the Examples described herein: QuantumTune Master V 1.2 or newer, Xcalibur V 1.4 SR1 or newer, TSQ Quantum 1.4or newer, and LCQuan V 2.0 with SP1 or newer. Liquid solvent/analyteexiting the analytical column flowed to the heated nebulizer interfaceof the MS/MS analyzer. The solvent/analyte mixture was converted tovapor in the heated tubing of the interface. Analytes in the nebulizedsolvent were ionized by ESI.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of 377.2±0.50 m/z. Ions entering quadrupole 2 (Q2)collided with argon gas to generate ion fragments, which were passed toquadrupole 3 (Q3) for further selection. Simultaneously, the sameprocess using isotope dilution mass spectrometry was carried out with aninternal standard, ¹³C, ¹⁵N₂-vitamin B2. The following mass transitionswere used for detection and quantitation during validation on positivepolarity.

TABLE 1 Mass Transitions for vitamin B2 and ¹³C, ¹⁵N₂-vitamin B2(Positive Polarity) Analyte Precursor Ion (m/z) Product Ion (m/z)vitamin B2 377.2 243.2 ¹³C, ¹⁵N₂-vitamin B2 380.2 246.2

Exemplary chromatograms for vitamin B2 and ¹³C, ¹⁵N₂-vitamin B2(internal standard) are found in FIGS. 1A-B.

Example 4 Intra-Assay and Inter-Assay Precision and Accuracy

Four quality control (QC) pools covering the reportable range of theassay were prepared from blank human serum (Biocell Laboratories, Inc.,Cat. No. 1131-00) spiked with vitamin B2 to a concentration of 12.5, 25,50, and 100 nmol/L.

Ten aliquots from each of the four QC pools were analyzed in a singleassay to determine the coefficient of variation (CV (%)) of a samplewithin an assay. Results are found in Table 2. All accuracies are withinan acceptable range of 85% to 115%.

TABLE 2 Intra-Assay Variation and Accuracy for vitamin B2 Level I LevelII Level III Level IV Sample (12.5 nmol/L) (25 nmol/L) (50 nmol/L) (100nmol/L) 1 12.1 26.8 49.0 97.0 2 14.2 26.2 51.0 101.7 3 11.0 27.4 45.596.9 4 14.7 22.9 46.5 96.7 5 10.0 27.1 48.0 94.7 6 14.0 26.8 48.6 92.0 712.9 23.3 48.3 97.8 8 12.0 24.6 47.6 96.8 9 12.7 26.0 47.7 98.1 10  13.627.4 50.7 93.1 Mean 12.7 25.9 48.3 96.5 (nmol/dL) Standard 1.5 1.7 1.72.7 Deviation (nmol/dL) CV (%) 11.7% 6.5% 3.5% 2.8% Accuracy 101.7%103.4% 96.6% 96.5% (%)

Ten aliquots of each of the same four QC pools were assayed over fivedays to determine the coefficient of variation (CV (%)) between assays.Results are found in Table 3. All accuracies are within an acceptablerange of 85% to 115%.

TABLE 3 Inter-Assay Variation and Accuracy Mean Measured Value Level ILevel II Level III Level IV Day (12.5 nmol/L) (25 nmol/L) (50 nmol/L)(100 nmol/L) 1 12.6 20.2 50.0 102.5 2 11.9 23.5 48.4 96.8 3 13.6 27.155.2 102.9 4 12.1 27.4 45.5 96.9 5 11.0 23.0 51.9 92.8 Mean 12.4 25.049.2 98.9 (nmol/dL) Standard 1.1 2.1 3.2 6.4 Deviation (nmol/dL) CV (%)8.6 8.6 6.5 6.5 Accuracy 99.5 99.9 98.4 98.9 (%)

Example 5 Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) andLimit of Detection (LOD)

The LLOQ is the point where measurements become quantitativelymeaningful. The analyte response at this LLOQ is identifiable, discreteand reproducible with a precision of greater than 20%. The LLOQ wasdetermined by assaying ten duplicates of blank human serum samplesspiked with vitamin B2 to concentrations of 2.5, 5, 12.5, and 25 nmol/L,then determining the reproducibility. Analysis of the collected dataindicates that samples with concentrations of about 5 nmol/mL or abovehad CVs less than 20%, while samples with concentrations lower than 5nmol/L had CVs greater than 20%. Thus, the LLOQ of this assay wasdetermined to be 5 nmol/L. The collected data is shown plotted in FIG.2.

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three standarddeviations from the zero concentration. To determine the LOD for thevitamin B2 assay, six replicates of blank human serum were assayed, andthe results analyzed. The LOD for this assay of 2.7 nmol/L.

Example 6 Vitamin B2 Reportable Range and Linearity

To establish the linearity of vitamin B detection in the assay, on twodifferent days three separate assays were conducted utilizing threechannels of the multiplexed TFLC system. Each assay included sevenspiked blank serum standards at concentrations ranging from 7.5 nmol/Lto 150.0 nmol/L. Linear regressions were calculated for each assay,yielding correlation coefficients of 0.9981 or greater (see Table 4,below). Graphs showing the linearity of the data are shown in FIG. 3.

TABLE 4 Fitting parameters for linearity studies Goodness of Fit AssayFitting Parameters (R²) Column 1 (Day 1) Y = 0.00745235 + 0.00916646 * X0.9987 Column 2 (Day 1) Y = 0.0310187 + 0.00827027 * X 0.9981 Column 3(Day 1) Y = 0.00721216 + 0.0089436 * X 0.9999 Column 1 (Day 2) Y =0.0182602 + 0.00791278 * X 0.9995 Column 2 (Day 2) Y = 0.0104523 +0.00771775 * X 0.9988 Column 3 (Day 2) Y = 0.0170813 + 0.00763554 * X0.9981

Example 7 Matrix Specificity

Matrix specificity was evaluated using human stripped serum and in-houseprepared blank EDTA plasma specimens. Samples of both matrices spiked toseven concentration levels ranging from 7.5 nmol/L to 150.0 nmol/L wereanalyzed over three days and the results compared. The study indicatedthat both matrices could be used for the assay. The results of thesestudies are presented in FIGS. 4A-C.

Example 8 Interference Studies

Eighteen drugs or compounds were investigated on their possibleinterference on vitamin B2 in this assay. These drugs are acetylsalicylic acid, 4-acetamidophenol, amiodarone, N-desethyl amidorone,felbamate, hydroxyzine hydrochloride, mycophenolic acid, flunitrazepam,fluoxetine, tacrolimus, rapamycin, cyclosporine, flecamide, propafenone,clozapine, norclozapine, lidocaine, and sirolimus. These drugs weremixed and spiked at a concentration of 50 ng/mL each to plasma samplescontaining vitamin B2 at three concentration levels (25 nmol/L, 50nmol/L, and 100 nmol/L). The samples were assayed together with vitaminB2 plasma samples with no drug additives, and results were compared. Thepercentage yielded from the drug additive versus the non-additivesamples were 90%, 88%, and 92% for the three concentration levels,respectively.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount of vitamin B2 in a biological sample when taken from a patient, said method comprising: (a) subjecting the sample, purified by turbulent flow liquid chromatography (TFLC) and high performance liquid chromatography (HPLC), to ionization under conditions suitable to produce one or more ions detectable by mass spectrometry; (b) determining the amount of said one or more ions by tandem mass spectrometry; and (c) using the amount of said one or more ions to determine the amount of vitamin B2 in the sample wherein said methods do not include addition of a protein precipitating agent to said sample prior to ionization.
 2. The method of claim 1, wherein said TFLC, HPLC, and tandem mass spectrometry are conducted with on-line processing.
 3. The method of claim 1, wherein said biological sample comprises plasma or serum.
 4. The method of claim 1, wherein said one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass to charge ratio of 377.2±0.5 and 243.2±0.5.
 5. The method of claim 1, wherein said one or more ions detectable by mass spectrometry comprise a parent ion with a mass to charge ratio of 377.2±0.5 and a fragment ion with a mass to charge ratio of 243.2±0.5.
 6. The method of claim 1, wherein the step of relating the amount of one or more ions detected by mass spectrometry to the presence or amount of vitamin B2 in the sample comprises comparison to an internal standard.
 7. The method of claim 6, wherein said internal standard is ¹³C, ¹⁵N₂-vitamin B2.
 8. The method of claim 1, wherein said method has a lower limit of quantitation within the range of 5 nmol/L and 25 nmol/L, inclusive.
 9. A method for determining the amount of vitamin B2 in a sample when taken from a patient, said method comprising: (a) purifying a biological sample, comprising: (i) subjecting the sample to an extraction column under turbulent flow conditions whereby vitamin B2 is retained by the extraction column; (ii) washing the extraction column with a first mobile phase under conditions whereby non-retained materials from the sample are washed from the column; (iii) applying a second mobile phase to said extraction column, whereby retained vitamin B2 is eluted from said extraction column; and (iv) applying vitamin B2 eluted from said extraction column to a high performance liquid chromatography column; (b) subjecting vitamin B2 from said sample to an ionization source under conditions suitable to produce one or more ions detectable by mass spectrometry; (c) determining the amount of said one or more ions by tandem mass spectrometry; and (d) using the amount of said one or more ions to determine the amount of vitamin B2 in the sample wherein said methods do not include addition of a protein precipitating agent to said sample prior to ionization.
 10. The method of claim 9, wherein said extraction column comprises particles with an average diameter greater than about 50 μm.
 11. The method of claim 9, wherein said sample comprises plasma or serum.
 12. The method of claim 9, wherein said one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass to charge ratio of 377.2±0.5 and 243.2±0.5.
 13. The method of claim 9, wherein said one or more ions detectable by mass spectrometry comprise a parent ion with a mass to charge ratio of 377.2±0.5 and a fragment ion with a mass to charge ratio of 243.2±0.5.
 14. The method of claim 9, wherein the step of relating the amount of one or more ions detected by mass spectrometry to the presence or amount of vitamin B2 in the sample comprises comparison to an internal standard.
 15. The method of claim 9, wherein said method has a lower limit of quantitation within the range of 5 nmol/L and 25 nmol/L, inclusive. 